The present invention concerns novel proteins capable of binding p35nck5a, and method of use of same.
As discussed in Ching Y P et al. (xe2x80x9cCloning of three novel neuronal Cdk5 activator binding proteins.xe2x80x9d, Gene. Jan. 25, 2000;242(1-2):285-94; PMID: 10721722), p35nck5a is a 35 kDa neuronal-specific protein from which is derived a 25 kDa activator protein named p25nck5a which, together with a catalytic subunit named Cdk5 (cyclin dependent kinase 5), forms an active kinase named neuronal cdc2-like kinase (Nclk), which is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics (Lew J et al., xe2x80x9cNeuronal cdc2-like kinase.xe2x80x9d, Trends Biochem Sci. January 1995;20(1):33-7; PMID: 7878742). Cdk5 itself exists in three distinct states:
i) a free monomer;
ii) a form in association with p25nck5a; and
iii) a form complexed with p35nck5a (Lee K Y et al., J Biol Chem. Jan. 19, 1996; 271(3):1538-43; PMID: 8576150).
The free monomeric form can be activated by bacterially expressed p25nck5a, whereas the p25nck5a-associated Cdk5 possesses endogenous kinase activity, and cannot be further activated. The form complexed with full length p35nck5a demonstrates no enzymatic activity even when exogenously activated, suggesting that the Cdk5 activity is suppressed. Further characterization shows that the p35nck5a/Cdk5 complex is a macromolecular complex of over 600 kDa in size. The simplest level of regulation may be revealed by the fact that Cdk5 is present in excess of its activators. Nck5a is thus essentially acting as the limiting factor for Cdk5 activity. However, it is still a mystery with regard to the functional difference between the intact and truncated activators. Therefore, it is very useful to uncover the identities of the various binding proteins in the macromolecular protein complex.
With regard to Cdk5, as well as being able to associate with p25nck5a, it can also associate with cyclin D and cyclin E but no kinase activity can be demonstrated. While Nck5a contains a very limited sequence homology to members of the cyclin family, evidence has shown that the structure of p25nck5a acquires a folding similar to that of cyclin. Northern analysis has indicated that Nck5a as well as its isoform, p39, are confined in neuronal tissue. Not only has Cdk5 adapted a distinct activator, but the regulation of the Cdk5 kinase activity is also considerably different from other CDKs. For instance, Cdk5 can be fully activated by association with the activator subunit alone, independent of the phosphorylation by Cdk-activating protein kinase (CAK), which is essential for most CDKs to achieve maximum activation. Furthermore, none of the known Cdk inhibitors has been demonstrated to inhibit Cdk5 kinase activity.
Thus proteins which are associated with Nclk, particularly those which bind to it or its subunits (including p35nck5a and Cdk5) and which may therefore affect (i.e. regulate) its activity are of particular interest and use. It has previously been shown (Qi Z et al., xe2x80x9cAssociation of neurofilament proteins with neuronal Cdk5 activator.xe2x80x9d, J Biol Chem. Jan. 23, 1998;273(4):2329-35; PMID: 9442078) that neurofilament is one of the p35nck5a associated proteins.
As discussed above, proteins which are associated with Nclk, particularly those which bind to it or its subunits and which may therefore affect (i.e. regulate) its activity, are of particular interest and use. The present inventors have now succeeded in isolating three novel p35nck5a associated proteins which are p35nck5a binding proteins, useful for a variety of purposes as detailed below, particularly as tools to analyse how p35nck5a interacts with other proteins.
Thus the present invention provides a p35nck5a binding protein comprising essentially the sequence of any one of the group consisting of SEQ ID NOs: 2, 4 and 6.
Also provided is a method to screen a large number of molecules or compounds for their ability to form complexes with the p35nck5a binding protein of the invention.
Also provided is a method of using the p35nck5a binding protein of the invention to purify a molecule or compound which specifically binds the p35nck5a binding protein of the invention.
Also provided is a method of determining the binding interaction of p35nck5a with a molecule or compound.
In particular, molecules or compounds (above) are chosen from the group consisting of peptides, agonists, antagonists, inhibitors, antibodies and pharmaceutical agents.